DNA methylation dynamics during epigenetic reprogramming of medaka embryo

Abstract

Post-fertilization epigenome reprogramming erases epigenetic marks transmitted through gametes and establishes new marks during mid-blastula stages. The mouse embryo undergoes dynamic DNA methylation reprogramming after fertilization, while in zebrafish, the paternal DNA methylation pattern is maintained throughout the early embryogenesis and the maternal genome is reprogrammed in a pattern similar to that of sperm during the mid-blastula transition. Here, we show DNA methylation dynamics in medaka embryos, the biomedical model fish, during epigenetic reprogramming of embryonic genome. The sperm genome was hypermethylated and the oocyte genome hypomethylated prior to fertilization. After fertilization, the methylation marks of sperm genome were erased within the first cell cycle and embryonic genome remained hypomethylated from the zygote until 16-cell stage. The DNA methylation level gradually increased from 16-cell stage through the gastrula. The 5-hydroxymethylation (5hmC) levels showed an opposite pattern to DNA methylation (5-mC). The mRNA levels for DNA methyltransferase (DNMT) 1 remained high in oocytes and maintained the same level through late blastula stage and was reduced thereafter. DNMT3BB.1 mRNA levels increased prior to remethylation. The mRNA levels for ten-eleven translocation methylcytosine dioxygenases (TET2 & TET3) were detected in sperm and embryos at cleavage stages, whereas TET1 and TET3 mRNAs decreased during gastrulation. The pattern of genome methylation in medaka was identical to mammalian genome methylation but not to zebrafish. The present study suggests that a medaka embryo resets its DNA methylation pattern by active demethylation and by a gradual remethylation similar to mammals.

Keywords: DNA methylation; Epigenetic reprogramming; medaka embryo.

Figure 1.

Genome methylation (5-mC) and hydroxymethylation (5-hmC) in medaka during embryogenesis. (a): Constitutive global DNA methylation; one-way ANOVA analysis showed p < 0.001; two-sample t-test showed significant differences between Sperm vs. Oocyte, Sperm vs. Zygote and Late gastrula vs. Early neurula; (b): constitutive global DNA hydroxymethylation levels; one-way ANOVA analysis showed p < 0.0001, Tukey’s multiple comparisons test showed significant differences between Zygote, 2-cell, 4-cell vs. Sperm, blastula stages, and gastrula stages; (c): correlation between 5-mC and 5-hmC levels during embryogenesis. (d): differentially methylated probes between Sperm and Blastula embryos. Data represent mean ± SEM in Figure 1(a) and (b). Asterisk indicates statistical significance (*p < 0.05; **p < 0.01).

Figure 2.

DNA methyltransferases expression during embryogenesis measured by real-time qRT-PCR. Gene expressions in all the embryonic stages were normalized against expression in sperm (set as 1), RQ: Relative quantification. (a): DNMT1, one-way ANOVA analysis showed p < 0.0001, Tukey’s multiple comparisons test showed significant differences between Oocyte to blastula stages vs. Sperm, gastrula stages and Early neurula. (b): DNMT3AA, one-way ANOVA analysis showed no significant differences among examined stages. (c): DNMT3BA, one-way ANOVA analysis showed p < 0.0001, Tukey’s multiple comparisons test showed significant differences between Early gastrula vs. all other examined stages. (d): DNMT3BB.1, one-way ANOVA analysis showed p < 0.0001, Tukey’s multiple comparisons test showed significant differences between 16-cell, Late blastula vs. Sperm, Oocyte, Zygote, Mid gastrula, Late gastrula, and Early neurula. (e): A relationship between DNMT3BB.1 expression and global 5-mC levels. Data represent mean ± SEM. Asterisk indicates statistical significance (**p < 0.01, ***p < 0.001).

Figure 3.

TET methylcytosine dioxygenases expression during early embryonic development measured by real-time qRT-PCR. Gene expressions in all the embryonic stages were normalized against expression in sperm (set as 1). RQ: Relative quantification. (a): TET1, one-way ANOVA analysis showed p < 0.0001, Tukey’s multiple comparisons test showed significant differences between 16-cell, Late morula vs. Sperm, Oocyte, Zygote, blastula stages, gastrula stages, and Early neurula. (b): TET2, one-way ANOVA analysis showed p < 0.0005, Tukey’s multiple comparisons test showed significant differences between Sperm vs. Mid gastrula, Late gastrula, and Early neurula. (c): TET3, one-way ANOVA analysis showed no significant differences among examined stages. TET1, TET2, and TET3 are paternally expressed and TET2 expression began after genome-zygotic transition. TET3 expression was absent after genome-zygotic transition. Data represent mean ± SEM. Asterisk indicates statistical significance (**p < 0.01, ***p < 0.001).

Figure 4.

DNA methylation reprogramming model during embryogenesis in medaka. (a): Sperm was hypermethylated and oocyte was hypomethylated. Paternal genomic methylation was erased in zygote (Step 1). Embryos stayed in hypomethylation status during first several cell cycles (Step 2). Global DNA methylation levels increased from 16-cell stage to Late blastula stage (Step 3). Embryos maintained hypermethylation during gastrula stages (Step 4). Global DNA methylation level decreased from gastrula to neurula stage. Relative quantification expression of genes involved in DNA methylation and hydroxymethylation were showed on lower panel. (b): A comparative epigenetic programming during embryogenesis in three model species – zebrafish [29,30], medaka, and mice [23,26].